The present invention relates to the expression of DNA, genes, cDNAs, proteins, peptides and parts thereof in the excretory canal of the nematode worm C. elegans. In particular, the invention relates to promoter sequences which are capable of directing tissue-specific gene expression in the excretory canal of C. elegans, to expression vectors containing the promoter sequences, to transgenic C. elegans specifically expressing reporter genes in the excretory canal, to methods of identifying chemical agents that affect the morphology of the excretory canal and to use of these agents in the pharmacological treatment of diseases for which the C. elegans excretory canal serves as a model.
The C. elegans Excretory Cell
The excretory system of the nematode C. elegans consists of three cells: a single large excretory cell, a duct cell and a pore cell that interfaces with the duct to the main body hypodermis. The excretory cell is the largest mononucleate cell in C. elegans. The nucleus and cell body of the excretory cell is situated at the terminal bulb of the pharynx. The cell itself is shaped in an H-form, with the two arms situated along the lateral lines for almost the entire length of the worm, and slightly dorsal. The excretory cell is polarized, having an apical domain facing the lumen of the excretory canal and a basal domain facing outside. The structure and the organization of the C. elegans excretory system suggest that it may be used for osmoregulation and can therefore be considered as a model for the vertebrate nephron.
Various mutant C. elegans have been reported which have an aberrant phenotype in the excretory canal. These aberrant phenotypes include cyst formation, short canals and branched canals. Various mutations affecting the excretory canal can be traced back in C. elegans II, ed. Riddle, Blumenthal, Meyer and Priess, Cold Spring Harbor Laboratory Press, 1997.
Drug Discovery in Growth Cone Steering.
Regulation of cell motility, cell shape and the outgrowth of axons or other cell outgrowths are all essential processes in the morphogenesis and function of both unicellular and multicellular organisms. Furthermore, the control of these processes is disturbed in a variety of diseases in which receptors, extra-cellular signals and intra-cellular pathways are over- or under-stimulated. The discovery of new genes, proteins and peptides that are involved in these processes and chemical entities which modulate them would very much help the understanding of these processes. Accordingly, there is a need to develop new methods for the discovery of novel molecules involved in the cell motility, cell shape and cell outgrowth process, and to establish their function. In addition, since malfunction of these biological processes can lead to disease there is also a need to discover chemical entities which modulate these processes which may be useful as pharmaceuticals. Diseases associated with cell motility, cell shape and cell outgrowth include cancerous disease, more particularly tumor formation, tumor metastasis and vascularisation of tumors.
Drug Discovery in Renal Diseases.
In the drug discovery process it is established practice to develop a model of a disease which can be used in the development of assays to screen for compounds with potential pharmaceutical activity. For kidney diseases, and more specifically kidney cyst formation, two different types of disease models currently exist; models based on cell cultures of renal epithelial cells and mouse models. Although these systems have been presented as models for cystic diseases, such as autosomal dominant polycystic kidney disease (ADPKD), they have several disadvantages.
The models based on cell cultures can never be compared with a live multicellular organism. Where aberrant growth indicative of cyst formation has been observed in cultures of different cells, it has proven difficult to develop efficient compound screens from these models. Furthermore, even if chemicals can be discovered that modulate cell growth and hence cyst formation in culture, it remains difficult to prove that these compounds will have analogous effects in the renal systems of multicellular organisms.
The developed mouse models for renal cyst diseases have the disadvantage that they are not suitable for middle to high throughput screening for the discovery of pharmacological compounds. Accordingly, there remains a clear need for an alternative model of renal diseases which more accurately models the renal systems of multicellular organisms but which is practical for use in middle to high throughput screening.
The present invention relates to the use of the C. elegans excretory cell in the drug discovery process. The C. elegans excretory canal is an efficient tool to study various developmental biological features; it is formed during the larval stages of the nematode and the canals are observed to grow along the animal in early development. Hence, the development of the excretory canal is an efficient tool to study growth cone steering and defects that might arise during its development and the excretory canal can be used as a model for the development of drug screens in the area of growth cone steering and directional outgrowth.
The C. elegans excretory cell and excretory canal can also be considered as a model of the human kidney nephron. The excretory canal has analogous apical-basal polarities as can be found in certain kidney cells and which are relevant for cellular function. Hence, studying the excretory canal may help to develop new tools against kidney diseases. Furthermore, the excretory canal can be used as a model for the development of drug screens in the area of kidney diseases.
In order to exploit the potential of the C. elegans excretory cell and excretory canal both as a disease model and in the development of drug screens it would be advantageous to be able to express any gene or cDNA of interest, including reporter genes, specifically in the excretory cell and excretory canal. To achieve this would require the identification of a tissue-specific promoter which is active in the excretory cell.
The present inventors have identified, through the use of biochemical, molecular biology and transgenic techniques, a promoter fragment that specifically directs transcription in the C. elegans excretory cell in a very efficient way. From this promoter fragment several deletions have been generated that still promote transcription, and hence gene expression, in the excretory cell of C. elegans. These promoter fragments are useful tools as they can be used to direct specific expression of any DNA fragment of interest in the excretory cell and excretory canal.
Accordingly, in a first aspect the invention provides a DNA fragment which is capable of functioning as a promoter directing gene expression in the excretory cell of C. elegans, which DNA fragment comprises the sequence of nucleotides set forth in any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or a fragment thereof in the absence of any other sequence of consecutive nucleotides from the C. elegans genome (i.e., an isolated DNA fragment).
According to another aspect of the invention, an isolated nucleic acid molecule, is provided. The isolated nucleic acid molecule can comprise: (a) nucleic acid molecules which hybridize under stringent conditions to a molecule consisting of a nucleic acid having a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, and which direct expression of a heterologous DNA fragment to the excretory canal of C. elegans, (b) deletions, additions and substitutions of (a) which direct expression of a heterologous nucleic acid to the excretory canal of C. elegans, and (c) complements of (a) or (b) which direct expression of a heterologous nucleic acid to the excretory canal of C. elegans. 
According to another aspect of the invention, an expression vector which is suitable for directing tissue-specific expression of a heterologous DNA fragment in the excretory cell of C. elegans is provided. The expression vector comprises a promoter, the promoter comprising a DNA fragment described above in the first aspect of the invention, positioned to direct expression of the heterologous DNA fragment. In one embodiment, the heterologous DNA fragment is a reporter gene. In certain embodiments, the reporter gene encodes green fluorescent protein, xcex2-galactosidase, xcex2-lactamase, luciferase, acetohydroxyacid synthase, alkaline phosphatase, xcex2-glucuronidase, chloramphenicol acetyltransferase, horseradish peroxidase, nopaline synthase or octapine synthase.
According to another aspect of the invention, a host cell transformed or transfected with any of the foregoing expression vectors, is provided. In important embodiments, the host cell is a C. elegans cell.
According to still another aspect of the invention, a transgenic C. elegans containing a transgene comprising a promoter which is capable of directing tissue-specific gene expression in the excretory cell of C. elegans operatively linked to a protein-encoding DNA fragment is provided. Preferred DNA fragments comprising the promoter are as described above. In important embodiments, the protein-encoding DNA fragment comprises a reporter gene encoding green fluorescent protein, xcex2-galactosidase, xcex2-lactamase, luciferase, acetohydroxyacid synthase, alkaline phosphatase, xcex2-glucuronidase, chloramphenicol acetyltransferase, horseradish peroxidase, nopaline synthase or octapine synthase. In certain embodiments, the transgene is stably integrated into a chromosome of the C. elegans. In some embodiments, the transgenic C. elegans further comprises a second transgene, wherein the second transgene comprises a promoter suitable for directing tissue-specific gene expression in the excretory cell of C. elegans operatively linked to a reporter gene. Preferred promoters of the second transgene are the same as those for the first transgene. Preferred reporter genes are also as described above. In further embodiments, one or both of the transgenes may be integrated into a chromosome of the C. elegans. 
According to another aspect of the invention, a method of identifying a mutation in a gene involved in growth cone steering, cell motility, cell shape, tumor formation, tumor metastasis, vascularisation of tumors, renal development, kidney disease, the development of the excretory canal, cytoskeletal organisation or surface to cytoskeleton signalling, is provided. The method involves contacting a transgenic C. elegans which expresses a reporter gene in the excretory canal with a mutagen, and screening for phenotypic changes in the excretory canal. In some embodiments, the mutagen is EMS, UV-TMP or X-rays. Preferred transgenic C. elegans are as described above. The transgenic C. elegans may be a wild-type strain or a selected mutant strain.
According to yet another aspect of the invention, a method of determining whether a compound is an inhibitor or an enhancer of the regulation of growth cone steering, cell motility, cell shape, tumor formation, tumor metastasis, vascularisation of tumors, renal development, pathways involved in kidney disease, development of the excretory canal, cytoskeletal organisation or surface to cytoskeleton signalling, is provided. The method involves contacting a sample of the compound with a transgenic C. elegans which expresses a reporter gene in the excretory canal, and screening for phenotypic changes in the excretory canal. Preferred transgenic C. elegans are as described in any of the foregoing aspects of the invention.
According to another aspect of the invention, a compound which is identifiable as an inhibitor or an enhancer of the regulation of growth cone steering, cell motility, cell shape, tumor formation, tumor metastasis, vascularisation of tumors, renal development, pathways involved in kidney disease, development of the excretory canal, cytoskeletal organisation or surface to cytoskeleton signalling using the method of the invention described above in the immediately preceding paragraph, is provided. In certain embodiments, the compound is used as a medicament for promoting neuronal regeneration, re-vascularisation or wound healing. In some embodiments, the compound is used as a medicament for the treatment of chronic neuro-degenerative diseases, tumor formation, tumor metastasis, tumor vascularisation, kidney diseases, kidney cyst formation, polycystic kidney diseases, cell migration diseases or immunological diseases. In further embodiments, the compound is used to prepare a pharmaceutical composition. The pharmaceutical composition provided according to the invention, therefore, comprises the compound and a pharmaceutically acceptable carrier, diluent or excipient thereof.
According to still another aspect of the invention, a method of determining the function of a protein encoded by a DNA fragment, is provided. The method involves (a) providing a transgenic C. elegans containing a transgene comprising a promoter suitable for directing tissue-specific gene expression in the C. elegans excretory cell operatively linked to a protein encoded by a DNA fragment, and (b) observing any phenotypic changes in the excretory canal of the transgenic C. elegans. Preferred promoter sequnces, reporter genes, transgenes and transgenic C. elegans, are as described above.
According to still a further aspect of the invention, a method of determining whether a compound is a modulator of growth cone steering, cell shape, tumor formation, tumor metastasis, vascularisation of tumors, cell motility, renal development, a pathway involved in kidney disease, development of the excretory canal, cytoskeletal organisation or surface to cytoskeleton signalling, is provided. The method involves (a) contacting a sample of the compound with a transgenic C. elegans expressing a DNA fragment encoding a protein involved in the regulation of growth cone steering, cell shape, cell motility, renal development or a pathway involved in kidney disease, which transgenic C. elegans contains a transgene comprising a promoter which is suitable for directing tissue-specific gene expression in the C. elegans excretory cell operatively linked to the DNA fragment; and (b) screening for phenotypic changes in the excretory canal. Preferred promoter sequnces, reporter genes, transgenes and transgenic C. elegans, are as described above. In important embodiments, the transgenic C. elegans has an abnormal excretory canal phenotype. In certain embodiments the DNA fragment expressed in the excretory cell of the transgenic C. elegans rescues an abnormal excretory canal phenotype which is present in the genetic background of the transgenic C. elegans. 
According to another aspect of the invention, a compound which is identifiable as a modulator of growth cone steering, cell shape, cell motility, tumor formation, tumor metastasis, vascularisation of tumors, renal development, a pathway involved in kidney disease, development of the excretory canal, cytoskeletal organisation or surface to cytoskeleton signalling using the method described above in the immediately preceding paragraph, is provided. In certain embodiments, the compound is used as a medicament for promoting neuronal regeneration, re-vascularisation or wound healing. In some embodiments, the compound is used as a medicament for the treatment of chronic neuro-degenerative diseases, tumor metastasis, tumor formation, tumor vascularisation, kidney diseases, kidney cyst formation, polycystic kidney diseases, cell migration diseases or immunological diseases.
According to still a further aspect of the invention, a pharmaceutical composition comprising a compound as described in the immediately preceding paragraph, and a pharmaceutically acceptable carrier, diluent or excipient thereof, is provided.
According to yet another aspect of the invention, a method of identifying further components of a biochemical pathway on which a compound identifiable as a modulator of growth cone steering, cell shape, cell motility, renal development, a pathway involved in kidney disease, development of the excretory canal, cytoskeletal organisation or surface to cytoskeleton signalling may act, is provided. The method involves (a) providing a transgenic C. elegans which expresses a reporter gene in the excretory canal, (b) contacting the transgenic C. elegans with a mutagen, (c) contacting the mutated C. elegans with a compound which is identifiable as a modulator of growth cone steering, cell shape, cell motility, renal development, a pathway involved in kidney disease, development of the excretory canal, cytoskeletal organisation or surface to cytoskeleton signalling, and (d) screening for phenotypic changes in the excretory canal. Preferred mutagens, promoter sequnces, reporter genes, transgenes and transgenic C. elegans, are as described above.
These and other aspects of the invention, as well as various advantages and utilities, will be more apparent with reference to the drawings and detailed description of the preferred embodiments.